Method

ABSTRACT

The present invention relates to a method and apparatus for isolating a component from a fluid mixture, in particular for separating out or concentrating a discrete component of a fluid sample in a microchamber.

[0001] The present invention relates to a method and apparatus forisolating a component from a fluid mixture, in particular for separatingout or concentrating a discrete component of a fluid sample.

[0002] Numerous methods may be employed to separate particles fromsuspensions or to separate different types of particles from each other.Simple physical separation methods such as filtering or centrifugaltechniques are commonly used. More sophisticated methods may be employedto separate biological material (eg cells). For example, U.S. Pat. No.6,013,188 discloses a means for attaching magnetic material tobiological cells in a suspension and the use of an applied magneticfield to separate the modified cells from the suspension. Techniquesbased upon electrophoresis are frequently used as a means for separatingcells from one another.

[0003] WO-A-98/46985 (Payne et al) discloses a method for assessing thecomposition of a liquid sample using resonance impedance measurements.This enables small changes in bulk electrical properties of the sampleto be monitored and correlated with changes in the composition of theliquid.

[0004] The present invention is based on the recognition that theimpedance characteristics of a fluid sample which has been made part ofa resonant electrical circuit are a useful analytical or manipulativetool. In particular, the impedance characteristics may be used toisolate a component of the sample in a dynamic system.

[0005] Thus viewed from one aspect the present invention provides amethod for isolating a proportion of a fluid mixture (eg a discretecomponent such as cells or particles) from the fluid mixture as a whole,said method comprising the steps of:

[0006] allowing the fluid mixture to flow through a measuring chamber,

[0007] applying an electrical signal at one or more frequencies to theproportion of the fluid mixture in the measuring chamber;

[0008] measuring an impedance quantity characteristic of the proportionof the fluid mixture in the measuring chamber at the one or morefrequencies; and

[0009] causing the proportion of the fluid mixture to flow to a uniquedestination remote from the remainder of the fluid mixture when theimpedance quantity measured at the one or more frequencies attains apredetermined target (eg is at or near to or exceeds a predeterminedtarget level of the impedance quantity).

[0010] The method is particularly advantageous for separating out thecomponents of a very small volume of a fluid sample in a microchamberwhere the measured impedance quantity (eg spectrum) of that volume ofthe sample is a fingerprint ie characteristic of the proportion of thefluid in the microchamber.

[0011] In a preferred embodiment, the proportion of the fluid mixture isa discrete component such as cells or particles.

[0012] In a preferred embodiment, the one or more frequencies at whichthe impedance quantity is measured includes a resonant frequency. Theimpedance quantity may be the dissipation factor.

[0013] In a preferred embodiment, the electrical signal is applied ateach of a plurality of frequencies in a frequency range and theimpedance quantity is measured at each of the plurality of frequenciesin the frequency range. Preferably the plurality of frequencies includesa resonant frequency. By selecting individual frequencies in a frequencyrange, the amount of data handling may be reduced and the rate ofisolation increased. A suitable number of frequencies may be for example10. If desired, the plurality of frequencies in the range are sufficientin number to generate an impedance spectrum characteristic of theproportion of the fluid mixture in the measuring chamber. Typicallyfrequencies are selected over a broad frequency range eg 100 Hz to 2MHz.

[0014] In a preferred embodiment, the electrical signal is applied at asingle frequency and the impedance quantity is measured at thatfrequency as the fluid mixture flows through the measuring chamber. Theflow rate through the measuring chamber and hence the rate of isolationis generally governed by the time taken to record and process theimpedance quantity measurements. Measurements at a single frequency aremore rapidly obtained than those at a plurality of frequencies and sothe rate of isolation is advantageously optimised. Preferably, thesingle frequency is at or near to the resonant frequency of themeasuring chamber containing the proportion of the fluid mixture. Afrequency at or near to the resonant frequency provides the largestvariations in the measured impedance quantity and hence a more sensitivemethod.

[0015] Generally speaking, the volume of the measuring chamber isconsistent with the dimensions of the discrete component which it isdesired to isolate. In a preferred embodiment of the method of theinvention, the volume of the proportion of fluid mixture in themeasuring chamber is less than 10⁻⁶ litres, preferably less than 10⁻⁹litres, more preferably less than 10⁻¹² litres and even as low as about10⁻¹⁵ litres. A preferred range for the volume of the proportion of thefluid mixture in the measuring chamber is 10⁻⁶ to 10⁻¹⁵ litres.

[0016] Whilst the applicants do not wish to be bound by theoreticalconsiderations it is noted that by carrying out impedance measurementson very small proportions of fluid mixtures (eg nanolitre or smallervolumes), the fluid mixture as a whole cannot be considered as ahomogenous fluid mixture of components but rather a fluid mixture ofdiscrete components. By fabricating a measuring chamber of dimensionssimilar to those of the discrete components of interest, it is possibleto conduct electrical impedance measurements on the discrete componentsas they pass through the measuring chamber and use the results toisolate these components. In other words, the measured impedancequantity of the proportion of fluid mixture in the measuring chamber canbe considered to be largely characteristic of either a discrete cell orof the discrete fluid medium and the impedance quantity measured for acell and for the fluid medium will be quite distinct. Such impedancemeasurements and the instruments for carrying them out have beendescribed generally by Ayliffe et al, IEEE Journal of MicromechanicalSystems, 8, 1, 50-57, 1999.

[0017] In the method of the invention, the fluid mixture may comprisetwo or more liquids. Alternatively it may comprise a liquid (or mixtureof liquids) with one or more dissolved or suspended components. Thefluid mixture may comprise particles or cells suspended in a liquidmedium. For example, the fluid mixture may be a blood extract comprisingblood cells and liquid medium. Alternatively the fluid mixture may be asolution eg a polymer containing solution. Preferably the fluid mixtureis a binary system.

[0018] In a preferred embodiment of the method of the invention, thecomponent which it is desired to isolate from a fluid mixture isinitially calibrated in the measuring chamber by measuring its impedancequantity (the calibrated impedance quantity) at the one or morefrequencies. Preferably the calibrated impedance quantity is measured ata plurality of frequencies to obtain a spectrum (the calibratedimpedance spectrum). The calibrated impedance quantity (or spectrum) maybe stored in a memory device.

[0019] In a preferred embodiment of the method of the invention, theimpedance quantity of the proportion of the fluid mixture measured inthe measuring chamber is compared with the calibrated impedancequantity. Various steps may be employed for comparing a measuredimpedance quantity with a calibrated impedance quantity. For example,the measured impedance quantity may be compared with a database of knownimpedance quantities until an effective match is made. Alternatively,neural network techniques may be used for matching a known impedancequantity with a measured impedance quantity. Alternatively, asubtractive step may be used in which a known impedance quantity issubtracted from the measured impedance quantity. For example, in abinary system of biological cells and liquid media where the knownimpedance spectrum is that of the liquid media, if the measuredimpedance spectrum of the proportion of fluid mixture in the measuringchamber is characteristic of liquid media the spectra will cancel and ifthe measured impedance spectrum of the proportion of fluid mixture inthe measuring chamber is characteristic of cells the spectra will notcancel.

[0020] In a preferred embodiment of the method of the invention, thestep of causing the proportion of the fluid mixture to flow to a uniquedestination occurs when the impedance quantity measured at the one ormore frequencies is substantially the same as the calibrated impedancequantity at the one or more frequencies. Alternatively, the step ofcausing the proportion of the fluid mixture to flow to a uniquedestination occurs when the impedance quantity measured at the one ormore frequencies is above a predetermined threshold value.

[0021] In a preferred embodiment of the invention, on departing themeasuring chamber, the destination of the proportion of the fluidmixture (eg a discrete component) may be controlled so that it isdirected along a first path. Preferably, second and subsequentproportions of the fluid mixture (eg second and subsequent discretecomponents) are directed elsewhere (eg along second and subsequentpaths). For example the two components of a binary system may bedirected along discrete paths to two separate destinations.

[0022] In one embodiment of the method of the invention, the step ofcausing the proportion of the fluid mixture to flow to a uniquedestination remote from the remainder of the fluid mixture may becarried out by sending an appropriate signal to a separating means in oradjacent to the flow path when the predetermined target is attained. Theseparating means may be a deflecting means in the flow path. Forexample, the deflecting means may be one or more valves or a bafflesystem.

[0023] In an embodiment of the invention, the electrical signal is atime varying electrical signal. For example, the time varying electricalsignal may be periodic. Preferably, the time varying electrical signalis an alternating current (AC) signal. Preferably the electrical signalis a sine wave varying in either voltage or current. A means for varyingthe resonant frequency of the applied electrical signal may be used. Forexample, at least one inductor or one or more quartz crystal resonatorsmay be added in series or parallel. Conveniently, the means for varyingthe frequency of the applied electrical signal ensures that the resonantfrequency is below about 1 MHz. At such a resonant frequency, problemsassociated with instrumentation and digitisation are generally reduced.Alternatively higher frequencies (eg 500 MHz-1 GHz) may be used. As suchthis allows the parasitic inductances and capacitances to be used.

[0024] The measurement of the impedance quantity may comprise a time tofrequency domain transformation of the time varying electrical signal.The steps involved in such a measurement will be generally familiar tothose skilled in the art (see for example Perturbation Signals forSystem Identification, ed K Godfrey, Prentice Hill, 1993, UK). The timevarying electrical signal may be periodic and may comprise any suitablefunction or code eg a pseudo random binary sequence (PRBS), a Golaycode, a Walsh function, a Huffman sequence or any other suitable codedsequence. Other suitable signals, codes or methodologies such as whiteGaussian noise or wavelet analysis, impulse response or step responsemay be employed and will be generally familiar to those skilled in theart (see for example Signal Processing Methods for Audio Images andTelecommunications, ed P M Clarkson and H Stork, Academic Press, London,1995).

[0025] Viewed from a further aspect the present invention provides anapparatus for isolating a proportion of a fluid mixture (eg a discretecomponent such as cells or particles) from the fluid mixture as a whole,said apparatus comprising:

[0026] an electrical impedance measuring device adapted to permit thefluid mixture to flow through a measuring chamber, wherein said deviceis capable of measuring an impedance quantity characteristic of theproportion of the fluid mixture in the measuring chamber; separatingmeans in or adjacent to the flow path of the fluid mixture at or near tothe exit end of the measuring chamber; and

[0027] means for sending a signal to the separating means when theimpedance quantity attains a predetermined target whereby to cause theproportion of the fluid mixture to flow to a unique destination remotefrom the remainder of the fluid mixture.

[0028] The separating means may be a deflecting means in the flow path.For example, the deflecting means may be one or more valves or a bafflesystem.

[0029] In a preferred embodiment, the electrical impedance measuringdevice comprises an electrical signal applying means adapted to apply atime varying electrical signal to the measuring chamber at one or morefrequencies in a frequency range (preferably including a resonantfrequency) and measuring means for measuring an impedance quantitycharacteristic of the proportion of the fluid mixture in a measuringchamber at the one or more frequencies in the frequency range. In anembodiment of the apparatus of the invention, the electrical signalapplying means is capable of applying an ac signal of variablefrequency. In an embodiment of the apparatus of the invention, theelectrical signal applying means is capable of applying a variableelectrical signal sine wave varying in either voltage or current. In anembodiment of the apparatus of the invention, the electrical signalapplying means is capable of applying a time varying electrical signalwhich is periodic. The electrical signal applying means may comprise ameans for varying the frequency of the electrical signal to apply theelectrical signal at a plurality of frequencies in a range including theresonant frequency. For example, the apparatus may further comprise atleast one inductor or at least one quartz crystal resonator.Conveniently, the means for varying the frequency of the electricalsignal is arranged so that the resonant frequency is below about 1 MHz.At such a resonant frequency, problems associated with instrumentationand digitisation are generally reduced.

[0030] The electrical signal applying means may comprise at least twoelectrodes (eg gold microelectrodes). Numerous electrode materials,sizes and configurations are suitable (as desired) for the preferredembodiment. In a preferred embodiment of the apparatus of the invention,the electrical signal applying means comprises one or moremicroelectrodes of the type generally or specifically disclosed inWO-A-99/60392 (Farfield Sensors Limited) or specifically claimedtherein.

[0031] In one embodiment, the measuring chamber is configured so thatmeasurement of impedance quantities may be conducted in an effective1-dimensional electric field. For example, the measuring chamber maytake the form of a microchamber or microchannel. For example, themeasuring chamber may have the dimensions 5×3×2 microns. Themicrochamber or microchannel may be lined with one or moremicroelectrodes in the manner described by Ayliffe (supra).

[0032] In an embodiment of the apparatus of the invention, the measuringmeans may comprise an impedance analyser. In an embodiment of theapparatus of the invention, the measuring means may be capable ofperforming a time to frequency domain transformation of the time varyingelectrical signal.

[0033] Fluid transport methodologies for achieving flow of the fluidmixture through the measuring chamber are familiar to those skilled inthe art. A suitable method involves capillary flow techniques.

[0034] The volume of the proportion of fluid mixture which may besubjected to the method of the invention will be generally determined bythe amount of fluid mixture which can flow through the electricalimpedance measuring device and be processed. Ayliffe et al (supra) havedemonstrated that a measuring chamber with a volume approaching 120 flmay be used. The volume of the proportion of fluid mixture capable ofbeing processed then depends upon the time taken to effect a measurementof the impedance quantity of the proportion of fluid mixture in themeasuring chamber. This varies depending upon the instrument used formeasuring the impedance quantity. For example, a Hewlett Packard 4192Aimpedance analyser is capable of conducting approximately 6 measurementsper second at a single frequency. In practice, it is desirable to have aflow rate slower than the minimum time taken to conduct a measurementthereby to prevent proportions of the fluid mixture (eg discretecomponents) flowing through the measuring chamber and avoidingmeasurement cycles. An HP 4192A analyser has a measurement rate whichpermits a volume of approximately 500 fl per second to be processed (120fl measuring cell volume×4 measurements per second). Using a fastermeasurement regime allows greater volumes to be processed.

[0035] In an embodiment of the apparatus, a plurality of electricalimpedance measuring devices and separating means acting in parallel maybe fed from a single source of fluid mixture. This would enable thevolume throughput to be advantageously increased.

[0036] In general, there will be a finite volume of fluid mixturebetween the measuring chamber and the separating means (eg switchingvalve) which directs the flow to the desired destination. In order tosynchronise the detection mechanism and the valve opening, it may beassumed that at a constant flow rate it will take a time (t) for theproportion of the fluid mixture in the measuring chamber to be flushedout and flow to the valve. Once the impedance quantity (eg spectrum) ofthe proportion of fluid mixture is measured in the measuring chamber,the control signal may be delayed by time t. As such, the opening andclosing of the valve will coincide with the arrival of the proportion ofthe fluid mixture which was in the measuring chamber t seconds ago. Inpractice, t may be reduced slightly to take into account the fact thatthe measurement may have been conducted on a part of the proportion ofthe fluid mixture (ie the proportion could already be leaving themeasuring chamber once the impedance measurement was made). As such, itwould be an advantage to open the valve slightly ahead of the proportionof fluid mixture to increase the effectiveness of isolation.

[0037] In a preferred embodiment of the method of the invention, thestep of causing the proportion of fluid mixture to flow to a uniquedestination may be repeated through secondary measuring chambers andseparating means to improve the effectiveness of isolation. Furthermeasuring chambers and separating means may be used if desired.Alternaively the proportion of fluid mixture may be recycled through thesame measuring chamber and separating means to optimise isolation.

[0038] In recognising that the impedance characteristics of a fluidsample which has been made part of a resonant electrical circuit are auseful analytical tool, other patentably significant aspects of theinvention have been envisaged.

[0039] Viewed from a further aspect the present invention provides amethod for screening a fluid medium for the presence of an analyte, saidmethod comprising:

[0040] allowing the fluid medium to flow through a measuring chamber;

[0041] applying an electrical signal at one or more frequencies to theproportion of the fluid mixture in the measuring chamber;

[0042] measuring an impedance quantity characteristic of the proportionof the fluid mixture in the measuring chamber at the one or morefrequencies; and

[0043] comparing the impedance quantity of the proportion of the fluidmedium measured in the measuring chamber with a known impedance quantityof the analyte.

[0044] The method is particularly advantageous for screening a verysmall volume of a fluid sample in a microchamber where the measuredimpedance quantity (eg spectrum) of that volume of the sample is afingerprint ie characteristic of the proportion of the fluid in themicrochamber.

[0045] Viewed from a yet further aspect the present invention provides amethod for quantifying the presence of an analyte in a fluid medium,said method comprising:

[0046] allowing the fluid medium to flow through a measuring chamber;

[0047] applying an electrical signal at one or more frequencies to theproportion of the fluid mixture in the measuring chamber;

[0048] measuring an impedance quantity characteristic of the proportionof the fluid mixture in the measuring chamber at the one or morefrequencies, and

[0049] activating an audible or visual response when the impedancequantity of the proportion of the fluid medium measured in the measuringchamber attains a predetermined target (eg is at or near to or exceeds apredetermined target level of the impedance quantity).

[0050] The method is particularly advantageous for “counting” an analytein a very small volume of a fluid sample in a microchamber where themeasured impedance quantity (eg spectrum) of that volume of the sampleis a fingerprint ie characteristic of the proportion of the fluid in themicrochamber.

[0051] Preferably the audible or visual response is activated when themeasured impedance quantity exceeds the known impedance quantity of thecomponent of the fluid in which the analyte is suspended.

[0052] The present invention will now be described in a non-limitativesense with reference to the accompanying Figures in which:

[0053]FIG. 1 is a schematic representation of an embodiment of themethod and apparatus of the invention for separating an analyte from asolution.

[0054] In FIG. 1, there is represented an apparatus according to theinvention designated generally by reference numeral 1. A single sourceof a weak analyte (A) in solution 2 was fed by means of a pump 3 to anelectrical impedance measuring device 4. The electrical impedancemeasuring device 4 comprises a measuring chamber 5 together with ameasurement and control system 6. The measurement system may take theform of an HP4192A analyser. At the exit end of the measuring chamber,there is located a twin valve mechanism 7 which is responsive to signalsfrom the control system 6.

[0055] As the solution of analyte 2 is pumped through the measuringchamber 5, an impedance quantity is measured at one or more frequenciesof the electrical signal which is applied. The measured impedancequantity is then compared to a database containing the known impedancequantity of A by control system 6. When a match occurs, valve 7 a isclosed and valve 7 b is opened so that the component A alone passes fromthe measuring chamber to the beaker 8. When no match occurs, valve 7 ais opened is valve 7 b is closed so that components in the solution 2other than analyte A pass to the waste beaker 9.

1. A method for isolating a proportion of a fluid mixture from the fluid mixture as a whole, said method comprising the steps of: allowing the fluid mixture to flow through a measuring chamber; applying an electrical signal at one or more frequencies to the proportion of the fluid mixture in the measuring chamber; measuring an impedance quantity characteristic of the proportion of the fluid mixture in the measuring chamber at the one or more frequencies; and causing the proportion of the fluid mixture to flow to a unique destination remote from the remainder of the fluid mixture when the impedance quantity measured at the one or more frequencies attains a target impedance quantity.
 2. A method as claimed in claim 1 wherein the target impedance quantity is a calibrated impedance quantity.
 3. A method as claimed in claim 1 or 2 wherein the measuring chamber is a microchamber.
 4. A method as claimed in any preceding claim wherein the proportion of the fluid mixture is a discrete component selected from the group consisting of cells and particles.
 5. A method as claimed in any preceding claim wherein the one or more frequencies at which the impedance quantity is measured includes a resonant frequency.
 6. A method as claimed in any preceding claim wherein the impedance quantity is the dissipation factor.
 7. A method as claimed in any preceding claim wherein the electrical signal is applied at each of a plurality of frequencies in a frequency range and the impedance quantity is measured at each of the plurality of frequencies in the frequency range.
 8. A method as claimed in claim 7 wherein the plurality of frequencies includes a resonant frequency.
 9. A method as claimed in claim 7 or 8 wherein the plurality of frequencies in the range are sufficient in number to generate an impedance spectrum characteristic of the proportion of the fluid mixture in the measuring chamber.
 10. A method as claimed in any of claims 1 to 6 wherein the electrical signal is applied at a single frequency and the impedance quantity is measured at that frequency.
 11. A method as claimed in claim 10 wherein the single frequency is at or near to the resonant frequency of the measuring chamber containing the proportion of the fluid mixture.
 12. A method as claimed in any preceding claim wherein the volume of the proportion of fluid mixture in the measuring chamber is less than 10⁻⁶ litres.
 13. A method as claimed in any preceding claim wherein the fluid mixture is a binary system.
 14. A method as claimed in any preceding claim wherein the fluid mixture comprises two or more liquids.
 15. A method as claimed in any of claims 1 to 13 wherein the fluid mixture comprises a liquid (or mixture of liquids) with one or more dissolved or suspended components.
 16. A method as claimed in claim 15 wherein the fluid mixture comprises particles or cells suspended in a liquid medium.
 17. A method as claimed in either of claims 15 or 16 wherein the fluid mixture is a blood extract consisting essentially of blood cells and liquid medium.
 18. A method as claimed in any of claims 1 to 15 wherein the fluid mixture is a solution
 19. A method as claimed in claim 18 wherein the solution is a polymer containing solution.
 20. A method as claimed in claim 2 further comprising the initial steps of: allowing a calibrant fluid mixture to flow through a measuring chamber; applying an electrical signal at one or more frequencies to the proportion of the calibrant fluid mixture in the measuring chamber; measuring the calibrated impedance quantity of the proportion of the calibrant fluid mixture in the measuring chamber at the one or more frequencies; and correlating the calibrated impedance quantity of the proportion of the calibrant fluid mixture in the measuring chamber with a characteristic of the proportion of the calibrant fluid mixture in the measuring chamber, wherein the characteristic of the proportion of the calibrant fluid mixture in the measuring chamber is known.
 21. A method as claimed in claim 20 further comprising the step of: comparing the impedance quantity of the proportion of the fluid mixture measured in the measuring chamber with the calibrated impedance quantity.
 22. A method as claimed in any preceding claim comprising: causing the proportion of the fluid mixture to flow to a unique destination when the impedance quantity measured at the one or more frequencies is substantially the same as the target impedance quantity at the one or more frequencies.
 23. A method as claimed in any preceding claim comprising: causing the proportion of the fluid mixture to flow to a unique destination when the impedance quantity measured at the one or more frequencies is above the target impedance quantity.
 24. A method as claimed in any preceding claim further comprising: sending an appropriate signal to a separating means in or adjacent to the flow path when the target impedance quantity is attained.
 25. A method as claimed in any preceding claim wherein the electrical signal is a time varying electrical signal.
 26. An apparatus for isolating a proportion of a fluid mixture from the fluid mixture as a whole, said apparatus comprising: an electrical impedance measuring device adapted to permit the fluid mixture to flow through a measuring chamber, wherein said device is capable of measuring an impedance quantity characteristic of the proportion of the fluid mixture in the measuring chamber; separating means in or adjacent to the flow path of the fluid mixture at or near to the exit end of the measuring chamber; and means for sending a signal to the separating means when the impedance quantity attains a target impedance quantity whereby to cause the proportion of the fluid mixture to flow to a unique destination remote from the remainder of the fluid mixture.
 27. An apparatus as claimed in claim 26 wherein the separating means is a deflecting means in the flow path.
 28. An apparatus as claimed in claim 26 or 27 wherein the electrical impedance measuring device comprises: an electrical signal applying means adapted to apply a time varying electrical signal to the measuring chamber at one or more frequencies in a frequency range and measuring means for measuring an impedance quantity characteristic of the proportion of the fluid mixture in a measuring chamber at the one or more frequencies in the frequency range.
 29. An apparatus as claimed in claim 26, 27 or 28 wherein the electrical signal applying means comprises at least two microelectrodes.
 30. An apparatus as claimed in any of claims 26 to 29 wherein the measuring chamber is configured so that measurement of an impedance quantity is conducted in an effective 1-dimensional electric field.
 31. An apparatus as claimed in any claim 30 wherein the measuring chamber is a microchamber or microchannel.
 32. A method for screening a fluid medium for the presence of an analyte, said method comprising: allowing the fluid medium to flow through a measuring chamber; applying an electrical signal at one or more frequencies to the proportion of the fluid mixture in the measuring chamber; measuring an impedance quantity characteristic of the proportion of the fluid mixture in the measuring chamber at the one or more frequencies, and comparing the impedance quantity of the proportion of the fluid medium measured in the measuring chamber with a known impedance quantity of the analyte.
 33. A method for quantifying the presence of an analyte in a fluid medium, said method comprising: allowing the fluid medium to flow through a measuring chamber; applying an electrical signal at one or more frequencies to the proportion of the fluid mixture in the measuring chamber; measuring an impedance quantity characteristic of the proportion of the fluid mixture in the measuring chamber at the one or more frequencies; and activating an audible or visual response when the impedance quantity of the proportion of the fluid medium measured in the measuring chamber attains a target impedance quantity. 